This invention relates generally to protein tyrosine kinases and, more particularly, to Eph-related receptor tyrosine kinases and their manipulation for the control of cellular processes.
Receptor tyrosine kinases comprise a large family of proteins that share a number of structural features such as a glycosylated extracellular ligand-binding domain, a hydrophobic transmembrane domain and a conserved cytoplasmic catalytic domain. Integral membrane tyrosine kinases have been shown to mediate cellular signals important for growth and differentiation. The transduction of many extracellular signals to the cytoplasm occurs as a result of the binding of ligands such as growth factors, for example, to receptor tyrosine kinases at the cell surface. In most cases, ligand binding activates the cytoplasmic tyrosine kinase catalytic domain and culminates in tyrosine phosphorylation of multiple substrates in the cytoplasm.
Increased expression of membrane-spanning receptor tyrosine kinases frequently has been associated with alterations in normal cellular processes. The affected cellular processes include cell proliferation, differentiation and cancer, including, for example, human cancers. Specific examples of such cancers can include glioblastomas, squamous carcinomas and mammary carcinomas, which are associated with the amplification of the EGF receptor gene. Adenocarcinomas, breast cancers and gastric cancers similarly are associated with aberrant expression of the HER2/neu receptor and certain breast carcinomas overexpress the erbB-3 gene, for example.
The correlation between aberrant expression and transforming ability also extends to members of the Eph subclass of receptor tyrosine kinases. For example, carcinomas of the liver, lung, breast and colon show elevated expression of Eph. Unlike many other tyrosine kinases, this elevated expression can occur in the absence of gene amplification or rearrangement. Such involvement of Eph in carcinogenesis also has been shown by the formation of foci of NIH 3T3 cells in soft agar and of tumors in nude mice following overexpression of Eph. Moreover, an antigen present on the surface of a pre-B cell leukemia cell line also has been identified as a member of the Eph subclass. Wicks et al., Proc. Natl. Acad. Sci., USA 89:1611-1615 (1992). This leukemia- specific marker, termed Hek, appears to be similar to the chicken Cek4 and mouse Mek4 of the Eph subclass of receptor tyrosine kinases (see Sajjadi et al., The New Biologist 3:769-778 (1991), which is incorporated herein by reference). As with Eph, Hek also was overexpressed in the absence of gene amplification or rearrangements in, for example, hemopoietic tumors and lymphoid tumor cell lines.
In addition to their roles in carcinogenesis, a number of transmembrane tyrosine kinases have been reported to play key roles during development. Examples include the mouse c-kit proto-oncogene and the Drosophila genes "sevenless" and "torso," which are involved in pattern formation. Consistent with this developmental role, many receptor tyrosine kinases other than those described above also have been shown to be developmentally regulated and predominantly expressed in embryonic tissues. Examples of these other tyrosine kinases include Cek1, which belongs to the FGF subclass, and the Cek4 and Cek5 tyrosine kinases (Pasquale et al., Proc. Natl. Acad. Sci., USA 86:5449-5453 (1989); Sajjadi et al. (1991); and Pasquale, E. B., Cell Regulation 2:523-534 (1991), all of which are incorporated herein by reference).
Eph was the first member of the Eph subclass of tyrosine kinases to be identified and characterized by molecular cloning (Hirai et al., Science 238:1717-1720 (1987)). The name Eph is derived from the name of the cell line from which the Eph cDNA was first isolated, the erythropoietin-producing human hepatocellular carcinoma cell line, ETL-1. The general structure of Eph is similar to that of other receptor tyrosine kinases and consists of an extracellular domain, a single membrane spanning region and a conserved tyrosine kinase catalytic domain. However, the structure of the extracellular domain of Eph, which comprises an immunoglobulin (Ig) domain at the amino terminus, followed by a cysteine-rich region and two fibronectin type III repeats in close proximity to the transmembrane domain, is completely distinct from that of previously described receptor tyrosine kinases. The juxtamembrane domain and carboxy-terminus regions of Eph also are unrelated to the corresponding regions of other tyrosine kinase receptors. Thus, the discovery of Eph defined a new subclass of receptor-type tyrosine kinases.
In addition to the isolation and characterization of Eph, other related tyrosine kinases now have been identified. Cek4 and Cek5 were identified by screening a chicken embryo cDNA expression library with anti-phosphotyrosine antibodies (Sajjadi et al. (1991) and Pasquale, E. B. (1991)). This method of identification was successful because Cek4 and Cek5 are expressed in embryonic tissues and have tyrosine kinase activity even when expressed as partial fragments in bacteria. Other Eph-related kinases that have been identified include Hek (Wicks et al. (1992)), Sek (Gilardi-Hebenstreit et al, Oncogene 7:2499-2506 (1992)), Eck (Lindberg and Hunter, Mol. Cell. Biol 10:6316-6324 (1990)), Elk (Lhotak et al., Mol. Cell. Biol. 11:2496-2502 (1991)) and Eek (Chan and Watt, Oncogene 6:1057-1061 (1991)). These tyrosine kinases were cloned using a variety of methods.
The number of existing Eph-related kinases is not known and cannot be predicted. However, the Eph subclass already represents the largest known subclass of receptor tyrosine kinases, comprising at least 10 distinct members. The kinases belonging to the Eph subclass are so classified because each includes features such as the amino terminal Ig domain, the cysteine-rich stretch and two fibronectin type III repeats in the extracellular domain, which are conserved within the Eph subclass. However, despite these common structural features, the overall amino acid sequences outside the catalytic domain are quite different, indicating that different members of the Eph subclass interact with distinct ligands and substrates and, thus, exert distinct functions. This notion is supported by the differential distribution of different Eph-related kinases in adult tissues.
There is no indication whether other Eph-related kinases exist and, if so, what their relationship is to the known Eph-related kinases. Nevertheless, despite similarities among the Eph-related receptor tyrosine kinases, each is different and, as such, functions in related but distinct cellular processes. For example, many members of the Eph subclass are expressed in the nervous system during development and thus are likely to be involved in nerve regeneration processes. The aberrant expression or uncontrolled regulation of any one of these receptor tyrosine kinases can result in different malignancies and pathological disorders. Therefore, the identification and characterization of novel transmembrane tyrosine kinases should provide important insights into the mechanisms underlying oncogenesis and cellular growth control pathways.
There thus exists a need to identify additional receptor tyrosine kinases and to manipulate them in order to diagnose pathological conditions and control cellular processes. The present invention satisfies this need and provides related advantages as well.